Deciphering structure-function relationships is the fundamental underpinning of our biomedical knowledge and provides the basis for our ability to create novel materials and drugs by knowledge-based design. Powerful tools aimed towards deciphering structure-function outcomes is germ-line genetic manipulation in mice. Enamel is a composite tissue with unique material properties that are owed to its biological fabrication. Ameloblast cells create an enamel protein matrix that serves to control crystallite habit and the organization of crystallite bundles. We hypothesize that the structure of enamel, from the nanoscale to the macroscale, is the outcome of the function(s) of critical domains within the ameloblastin protein. Ablating protein expression by aknock out shows the requirement for amelogenin and ameloblastin protein for normal enamel formation, although the loss of function does not provide insight into the underlying mechanism of protein action. The loss of ameloblastin disrupts ameloblast cell interactions with the matrix resulting in ameloblasts detaching from the forming matrix and re-entering the cell cycle. Here the process of enamel formation is so severely disrupted that the underlying function(s) of the ameloblastin protein in normal tissue formation is difficult to discern. We hypothesize that using a knock-in strategy will allow us to link the identity of a specific ameloblastin protein domain(s) to the function(s) it contributes to formation of the enamel tissue. We propose the use of homologous recombination coupled with protein engineering of a human ameloblastin minigene in order to modify the mouse genome and to decipher the function(s) that human ameloblastin domain(s) contribute to enamel biomineralization. Insights from similar work performed for the amelogenin protein suggests that the approach of knock-in gene targeting of a minigene preserves the qualitative and quantitative aspects of gene expression while permitting the use of an ameloblastin minigene designed to express an engineered ameloblastin protein that will allow insights into the function of the engineered protein. Functional changes will be measured by alteration to stereotypic enamel architecture and by analysis of the material properties of the enamel[unreadable] in the knock in condition compared to wild type animals. This experimental strategy will contribute significant information to functional genomics and to further understanding of the only ectoderm-derived biomineralized tissue in the vertebrate body. Preliminary data from this group on the use of a similar strategy using a knock-in engineered amelogeninminigene suggest that this approach will yield novel insights into the structure-function relationship for the ameloblastin protein, the second most abundant protein contributing to enamel organic matrix assembly and biomineralization. Humanizing rodent enamel will also yield a new animal model that would be useful to investigators exploring the most prevalent infectious disease of mankind, dental caries. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: The function(s) for the second most abundant protein of the forming mammalian enamel matrix is not known. Knocking out ameloblastin demonstrated that it plays an essential role, as enamel did not form in the absence of ameloblastin. Here, we map the function(s) of ameloblastin domains to the production of an enamel matrix required to control enamel biomineralization thus[unreadable] humanizing an animal model used to study caries, the most prevalent infectious disease of humankind. [unreadable]